Multiplexed, sensitive, and on-chip molecular diagnostic assays are useful in both clinical and research settings. Many detection strategies employ amplification schemes to achieve sensitivity by labeling surface or bead-bound targets with enzymes that turn over substrate into fluorescent or colorimetric molecules. Since a single target-binding event is reported by the enzymatic turnover of several substrate molecules, the strategy provides signal amplification. In standard amplification reactions such as the commercially available enzyme-linked immunosorbent assay (ELISA), these enzyme-assisted amplification reactions occur on microplates with net volumes on the order of 100 μl and are still considered the gold standard for protein detection. Recent studies have however been successful in further amplifying net signal and gaining up to three orders of magnitude increase in assay sensitivity by shrinking the reaction volume to concentrate the reaction products. Running reactions in nanoliter (nL, 1 nL=10−9 liters) to femtoliter (fL, 1 fL=10−15 liters) sized volumes such as microwells or droplets has led to significant increases in detection sensitivities. By examining thousands of reaction volumes, some of these assays have digitized signal output at the lower end of their calibration curves, enabling single-molecule detection of target-enzyme complexes.
To this end, researchers have explored a number of platforms for the creation and utilization of stable and monodisperse miniature reaction compartments. For example, femtoliter-sized microwells, which are large enough to hold a single 3-5 micron (also micrometer, μm, 1 μm=10−6 meters) diameter bead, have been fabricated using etched optical-fiber bundles or injection molding of polymers. In other systems, similarly sized bead-filled droplets have been arrayed on hydrophobic surfaces patterned with hydrophilic wells. Individual beads with target-enzyme complexes and the enzymatic substrate solution are then confined into the compartments and sealed using mechanical force or, in more recent work, inert fluorinated oil. Meanwhile, slightly larger, picoliter (pL, 1 pL=10−12 liters) to nanoliter-sized microwells and surfactant-stabilized droplets have been made using soft lithography and microfluidic techniques. In all of these platforms, the confined reaction volume provides significant increases in reaction sensitivity in comparison to reactions run in bulk.
It is apparent that both microwells and droplets have favorable characteristics applicable to carrying out biological assays. While microwells are physically immobilized and have well-defined boundaries dictated by the fabrication process, droplets provide a naturally aqueous environment to foster biological reactions. However, water droplets require introduction of a solid substrate (e.g. microsphere) if they are to be functionalized with biological moieties such as nucleic acids. Furthermore, liquid manipulation in and out of microwells and droplets can be challenging and often requires intricate fluidics.